The main goal of this proposal is to determine the circumstances under which autoreactive T cells that have avoided negative selection in the thymus can defeat regulatory CD4+Foxp3+ T cells in the periphery. In our opinion, to achieve this goal it is necessary to study the features of individual autoreactive and regulatory T cells in their natural environment. In addition, these cells must express different antigen receptors (TCRs) specific for both self and non-self antigens. For this purpose, we have recently established a distinctive in vivo model where all T cells express a polyclonal but restricted repertoire of TCRs (TCRmini mice). In this model the CDR3 regions of TCRs can be used to trace individual naove, effector, and regulatory T cells in different stages of autoimmune disease. Now we also report that we have developed two in vivo models where T cells expressing this TCR mini- repertoire develop systemic or organ-specific autoimmunity. Subsequently, our proposal will investigate how the balance between antigen specific autoreactive and regulatory Foxp3+ CD4+ T cells clones can be destroyed, leading to the onset of autoimmune responses. In Specific Aim 1 we propose to determine if natural and adaptive TR cells play a distinctive role during onset and progression of autoimmune disease. We also hypothesize that though some natural TR cells express autoreactive TCRs and may lose Foxp3 expression the participation of these disabled TR cells in natural autoimmune responses is insignificant. Our second Specific Aim will examine if clonal escape of autoreactive T cells is driven by a few clones with higher TCR affinity for self MHC/peptide complexes or is caused by intrinsically higher frequency of autoreactive T cells. Our objective will also be to reveal if individual autoreactive T cell clones differ in their resistance to suppression by TR cells. In our third Specific Aim we will investigate if antigen-experienced Foxp3+ TR cells exhibit improved suppressor ability compared to naove Foxp3+ cells, and whether their TCR repertoire is more oligoclonal than the TCR repertoire found on the antigen-inexperienced Foxp3+ cells. PUBLIC HEALTH RELEVANCE This proposal focuses on investigating the mechanisms of tolerance induction by natural and adaptive Foxp3+ regulatory T lymphocytes. These regulatory T lymphocytes supervise the function of other immune cells and control autoreactive T cells preventing onset of autoimmunity. Better understanding of the mechanisms that autoreactive cells utilize to override regulatory function of CD4+Foxp3+ cells is critical for the development of better therapeutic strategies to treat autoimmune diseases.